Syrian hamsters recapitulate the two major forms of infection with Leishmania donovani seen in man. After systemic infection, these animals develop a progressive disease that mimics the clinical-pathological features of human VL. However, hamsters infected in the skin control the infection locally, and acquire immunity to systemic challenge, akin to sub-clinically infected individuals who develop immunity against visceral disease. These two broad features prompted us to initiate studies to dissect the molecular immunopathogenesis of L. donovani infection in this unique animal model. In preliminary studies we found that during progressive disease in the hamster model of VL, a Type 1 T cell response is mounted, but is ineffective and accompanied by undetectable NOS2 expression. Furthermore, in contrast to mouse macrophages, IFN-gamma-activated hamster macrophages do not produce detectable NO and are unable to control intracellular infection. These distinctions between hamsters and mice provide the impetus to directly compare and contrast mechanisms of macrophage activation/deactivation in these species, with particular focus on the NOS2-arginase metabolic pathways. We will extend in vitro findings to in vivo proof-of-principle studies in the hamster, again with the focus on the role of the NOS2-arginase metabolic pathways in resistance to disease and protective immunity. We will approach the role of NOS2 and arginase in infection with L donovani from two opposite, but complementary directions. In Specific Aims 1 and 2 we will determine the role of NOS2 and arginase expression in the pathogenesis of visceral infection through in vitro studies of infected hamster and mouse macrophages, and in vivo studies of systemically infected hamsters. Specific Aim 1 will test the hypothesis that the impaired capacity of hamster macrophages to control L. donovani infection is a result of the hypo-responsiveness of NOS2 to IFN-gamma-mediated activation, and that this NOS2 hyporesponsiveness is mediated through transcriptional mechanism(s). Specific Aim 2 will test the hypothesis that the impaired NOS2 expression sets the stage for an arginase-dominated alternative activation or deactivation pathway in infected hamster or human macrophages, which leads to impaired parasite killing. In Specific Aim 3 we will determine if the control of primary cutaneous infection and protection against secondary visceral challenge following acquisition of immunity is mediated by a reversal of the default toward low NOS2 and high arginase expression. We hypothesize that NOS2-independent mechanisms contribute to resistance and we will break new ground in the discovery of a gene expression profile that characterizes resistance to primary and secondary infection in the hamster. These studies in the hamster will fill a knowledge gap by defining mechanisms of disease and immunity that are strikingly different from those of the murine model.